WNK1–OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy
Abstract
:1. Introduction
2. Results
2.1. The Expression of wnk1a–osr1a/osr1b Is Upregulated during Embryonic Angiogenesis
2.2. The Expression of wnk1a and osr1b Is Upregulated in Endothelial Cells, and WNK1 and OSR1 Are Upregulated in Hepatoma Cells during Tumor-Induced Angiogenesis
2.3. [tert] and [tert x p53-/-] Transgenic Fish Develops HCC at 15 and 30 dpf
2.4. Expression of wnk1a, osr1b and stk39 Is Increased, and ppp2r1ba and ppp2r1bb Are Decreased in HCC Formation in the [tert] and [tert x p53−/−] Transgenic Fish
2.5. Expression of wnk1a and osr1b Is Increased in HCC Formation in the [HBx,src,p53−/−,RPIA] Transgenic Fish
2.6. Endothelial Cells Promote Hepatoma Cell Migration via WNK1–OSR1 axis
2.7. PPP2R1A Acts as a Repressor of WNK1 in Stimulating Hepatoma Cell Migration
2.8. Combinational Therapy Targeting WNK1–OSR1 with Oligo-Fucoidan Attenuates HCC Formation in [HBx,src,p53−/−,RPIA] Transgenic Fish
2.9. Oligo-Fucoidan Decreases the Elevated Cell Senescence-Associated β-Galactosidase Activity in Tert Transgenic Fish Treated with WNK1–OSR1 Inhibitors
3. Discussion
4. Materials and Methods
4.1. Zebrafish Husbandry
4.2. Embryo Collection
4.3. Cell Culture
4.4. Xenotransplantation
4.5. Embryonic Angiogenesis
4.6. RNA Extraction
4.7. Reverse-Transcription and Quantitative Polymerase Chain Reaction (QPCR)
4.8. Transfection
4.9. Transwell Migration Assay
4.10. Western Blot Analysis
4.11. Oral Gavage
4.12. Hematoxylin and Eosin Stain
4.13. Detection of Senescence-Associated β-galactosidase (SA-β-gal) Accumulation
4.14. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fish Lines | Promoter | Expressed Gene | Phenotype |
---|---|---|---|
AB wild-type | - | - | control |
Tg(fli1:EGFP) | fli1 | EGFP | Green fluorescence in the vessels. |
Tg(fabp10a:HBx,src,RPIA;myl7:EGFP)xtp53zdf1/zdf | fabp10a (liver-specific promoter) | HBx, src, RPIA tp53zdf1/zdf | Green fluorescence in heart. Develops HCC at 5 months of age. |
Tg(fli1:wnk1a;myl7:EGFP) | fli1 | wnk1a | Green fluorescence in heart. Overexpressed wnk1a in the vessels. |
Tg(fli1:EGFP)xTg(fli1:CreERT2;myl7:EGFP)xTg(loxP-wnk1a-DsRed-loxP) | fli1 | wnk1a CreERT2 loxP-wnk1a-DsRed-loxP | Vessels-specific knockout the wnk1a by adding the RU486 activates CreERT2. |
Tg(fli1:EGFP)xTg(fli1:wnk1a) | fli1 | wnk1a, EGFP | |
Tg(fabp10a:tert;myl7:EGFP)xtp53zdf1/zdf | fabp10a (liver-specific promoter) | tert tp53zdf1/zdf | Green fluorescence in heart. Develops HCC at 15, 30 days of age. |
Tg(fabp10a:tert;myl7:EGFP) | fabp10a (liver-specific promoter) | tert | Green fluorescence in heart. Develops HCC at 15 days of age. |
Gene Name | Primer Name | Sequencing |
---|---|---|
STK39 | h-Q-stk39_F | 5′-CATGAGTCAGTGCAGCCATC-3′ |
h-Q-stk39_R | 5′-TGTGTTCTCCTCGGTTGACA-3′ | |
OXSR1 | h-Q-oxsr1_F | 5′-AAAGACCTTTGTTGGCACCC-3′ |
h-Q-oxsr1_R | 5′-AGGATCGTTCTGCAGTGTCA-3′ | |
PPP2R1B | h-Q-ppp2r1b_F | 5′-GTCCTGACTTTGCCCACTGT-3′ |
h-Q-ppp2r1b_R | 5′-GAACCAATCCCCACTTGCTA-3′ | |
PPP2R1A | h-Q-PPP2R1A_F | 5′-TGACTGTCGGGAGAATGTGA-3′ |
h-Q-PPP2R1A_R | 5′-GGGAGAGAGACCCATGATGA-3′ | |
stk39 | z-Q-stk39_F | 5′-TGGACACCTGCACAAAACTG-3′ |
z-Q-stk39_R | 5′-TCGTTTTCTTTGACCCTGCG-3′ | |
oxsr1a | z-Q-oxsr1a_F | 5′-AGGTGGCCATTAAACGCATC-3′ |
z-Q-oxsr1a_R | 5′-GCAACTTCATGACCAGCCAA-3′ | |
oxsr1b | z-Q-oxsr1b_F | 5′-CATCAAACGCATCAATCTGG-3′ |
z-Q-oxsr1b_R | 5′-CGGTCTTGTGTTCACCCTTT-3′ | |
ppp2r1ba | z-Q-ppp2r1baF | 5′-TGGCAACAGTTGAAGAGACG-3′ |
z-Q-ppp2r1baR | 5′-AGAGCCCACAAGCAGAGGTA-3′ | |
ppp2r1bb | z-Q-ppp2r1bbF | 5′-AGACTTGGAGGCTCTGGTCA-3′ |
z-Q-ppp2r1bbR | 5′-GGTCTCCCTGCTGTCTTCAG-3′ | |
ccne1 | z-ccne1_F | 5′-CATGCCAAGCAAGAAAGTGCTA-3′ |
z-ccne1_R | 5′-GTGCTGGGAACACCTTCAGT-3′ | |
cdk1 | z-cdk1_F | 5′-CTCTGGGGACCCCTAACAAT-3′ |
z-cdk1_R | 5′-CGGATGTGTCATTGCTTGTC-3′ | |
cdk2 | z-cdk2_F | 5′-GGGCACTTTTGACATGGAGT-3′ |
z-cdk2_R | 5′-GTGCTGGGAACACCTTCAGT-3′ | |
actin | z-actin_F | 5′-CTCCATCATGAAGTGCGACGT-3′ |
z-actin_R | 5′-CAGACGGAGTATTTGCGCTCA-3′ | |
il1β | z-Q-il1b_ | 5′-CGCTCCACATCTCGTACTCA-3′ |
z-Q-il1b_R | 5′-ATACGCGGTGCTGATAAACC-3′ |
Component | Volume per Reaction (μL) |
---|---|
5× iScript Reaction Mix | 4 |
iScript Reverse Transcriptase | 1 |
RNase-free water | variable |
RNA template(1 μg) | variable |
Total volume | 20 |
Component | Volume per Reaction (μL) |
---|---|
cDNA (dilute with RNase-free water) | 3.8 |
Primer (2.5 μΜ of forward and reverse primer) | 1.2 |
2X SYBR Green (Catalog #: 4385618, Thermo Scientific, Waltham, MA, USA) | 5 |
Temperature | Time | Cycle | |
---|---|---|---|
Hold stage | 95 °C | 3 min | 1 |
PCR stage | 95 °C | 1 s | 40 |
60 °C | 20 s | ||
Melt curve stage | 95 °C | 15 s | 1 |
60 °C | 1 min | ||
95 °C | 15 s |
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Hou, C.-Y.; Ma, C.-Y.; Lin, Y.-J.; Huang, C.-L.; Wang, H.-D.; Yuh, C.-H. WNK1–OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy. Int. J. Mol. Sci. 2022, 23, 12100. https://doi.org/10.3390/ijms232012100
Hou C-Y, Ma C-Y, Lin Y-J, Huang C-L, Wang H-D, Yuh C-H. WNK1–OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy. International Journal of Molecular Sciences. 2022; 23(20):12100. https://doi.org/10.3390/ijms232012100
Chicago/Turabian StyleHou, Chia-Ying, Chung-Yung Ma, Yu-Ju Lin, Chou-Long Huang, Horng-Dar Wang, and Chiou-Hwa Yuh. 2022. "WNK1–OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy" International Journal of Molecular Sciences 23, no. 20: 12100. https://doi.org/10.3390/ijms232012100